GSM7474615: cr 30m Rep3; Saccharomyces cerevisiae; MNase-Seq
Briefly, cells were fixed for 15 min with 1% formaldehyde. Glycine was added to quench the reaction at a final concentration of 125 mM. Cells were sedimented, washed twice with cold PBS and stored at –80ºC until use. Extracts for MNase digestion were resuspended in 1M sorbitol/50mM Tris HCl pH 7,5 and digested 1 h with 4.5 mg of zymoliase 20T (AmsBio 120491-1). Samples were washed first with solution I (1 M sorbitol, 20mM Tris HCl pH8, 1mM EDTA, 150mM NaCl) and then with solution I 0.1mM PMSF, suspended gently in solution II (20mM Tris HCl pH 8, 1mM EDTA, 0.15M NaCl, 0.1mM PMSF, 0.2 % Triton), and treated 20 min with different concentrations of MNase (SIGMA N3755). The reaction was then stopped by adding 3mM EDTA and 4mM Tis HCl. The spheroplasts were broken with 10% SDS. To revert crosslinking, samples were incubated for 90 min at 37ºC with proteinase K and then overnight at 65ºC. DNA was extracted from samples using a standard phenol-chloroform extraction and treated with RNase A. MNase digested samples enriched in mononucleosomes were loaded in a 1% agarose gel, and the DNA corresponding to mononucleosomes was purified with a DNA purification kit.